Dysregulation of CD177+ neutrophils on intraepithelial lymphocytes exacerbates gut inflammation via decreasing microbiota-derived DMF

ABSTRACT Neutrophils synergize with intestinal resident intraepithelial lymphocytes (IELs) to serve as the first-line defense and maintain intestinal homeostasis. However, the underlying mechanisms whereby neutrophils regulate IELs to inhibit intestinal inflammation are still not completely understood. Here, we found that depletion of neutrophils (especially CD177+ subset) caused expansion of colitogenic TCRγδ+CD8αα+ IELs, increased intestinal inflammation, and dysbiosis after dextran sulfate sodium exposure or Citrobacter rodentium infection in mice. scRNA-seq analysis revealed a pyroptosis-related gene signature and hyperresponsiveness to microbiota in TCRγδ+CD8αα+ IELs from colitic Cd177−/− mice. Microbiota-derived fumarate and its derivative dimethyl fumarate (DMF), as well as fumarate-producing microbiotas, decreased in the feces of colitic Cd177−/− mice. Elimination of dysbiosis by antibiotics treatment or co-housing procedure and DMF supplementation restrained TCRγδ+CD8αα+ IEL activation. Consistently, DMF significantly alleviated intestinal mucosal inflammation in mice through restricting gasdermin D (GSDMD)-induced pyroptosis of TCRγδ+CD8αα+ IELs. Therefore, our data reveal that neutrophils inhibit intestinal inflammation by promoting microbiota-derived DMF to regulate TCRγδ+CD8αα+ IEL activation in a GSDMD-mediated pyroptosis-dependent manner, and that DMF may serve as a therapeutic target for the management of intestinal inflammation.


Introduction
Neutrophils are the early responders to inflammatory signals and are crucial in immune regulation, pathogen clearance, and recruitment of immune cells in the gastrointestinal tract. 1,2 Dysfunction of neutrophils contributes to impaired intestinal immune homeostasis and aggravated mucosal inflammation when pathogens invade. The underlying mechanisms involved in the dysregulated neutrophils include proinflammatory cytokine burst, irreducible immune responses, and discordant immune cell functions. 3 CD177 (NB1 or PRV1) is a glycosylphosphatidylinositol-linked glycoprotein expressed exclusively in neutrophils, neutrophilic metamyelocytes, and myelocytes, which binds platelet endothelial cell adhesion molecule-1 and functions in modulating neutrophil transmigration. Previous study has demonstrated that CD177 + neutrophils play a protective role in the pathogenesis of inflammatory bowel diseases (IBD) through increased IL-22 production and bactericidal activity. 4 Among the immune cells that contribute to the maintenance of intestinal homeostasis, intraepithelial lymphocytes (IELs) play a crucial role in preserving the mucosal barrier intact, patrolling the epithelial layer, and synergizing with rushing neutrophils to initiate the timely primary defense against exogenous pathogens. 5 Especially, TCRγδ + IELs are essential for limiting epithelial penetration of resident commensal microbiota through antimicrobial effectors (i.e., RegIIIγ), which are dependent on intestinal epithelial cell (IEC)-intrinsic MyD88 signaling following dextran sulfate sodium (DSS)-induced mucosal damage, indicating a critical role of proper TCRγδ + IEL functions in maintaining host-microbiota homeostasis following acute mucosal inflammation. 6,7 Optimal spatial interactions and cooperation between neutrophils and IELs are essential for maintaining gut mucosal immune homeostasis in response to pathobiont challenges. Loss of the delicate immunoregulation of neutrophils and IELs compromises the intestinal barrier integrity and drives a series of pathogenic disorders in the gut, such as neutropenic enterocolitis, infections, and IBD. 8,9 Notably, accumulating activated CD8 + IELs have been found to be closely associated with the pathogenesis of celiac disease and correlated with gastrointestinal manifestations present in coronavirus disease 2019 (COVID-19) patients infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 10,11 However, the underlying mechanisms whereby neutrophils regulate IELs and collectively preserve the intestinal homeostatic status are still not fully understood. Here, we reported an unanticipated mechanism by which neutrophils restrain intestinal mucosal inflammation through regulating IEL functions, mediated by microbial metabolite dimethyl fumarate (DMF), and provided novel insights into manipulating IEL activation by DMF as a potential therapeutic approach for patients suffering intestinal inflammatory disorders, e.g., inflammatory bowel disease (IBD).

Experimental animals
Cd177 −/− mice were purchased from the KOMP at the University of California (Davis, California, USA), bred, and maintained in the animal facility of Tongji University School of Medicine. C57BL/ B6 wild type (WT) mice (6-8 weeks) used for the establishment of the DSS-induced colitis model were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China), which were also bred with Cd177 −/− mice to generated Cd177 +/+ mice and used for microbiota analysis as WT littermates of Cd177 −/− mice. For co-housing experiments, 3-week-old Cd177 −/− mice and WT littermates were co-housed for at least 4 weeks (co-housing) or still housed separately. 12 These mice were raised in an independent ventilation cage under specific pathogenfree conditions with a 12-hour light cycle and were fed autoclaved food and water. Male mice were used at 8-10 weeks of age with 20-25 g of body weight. All animal experiments in this study were reviewed and approved by the Institutional Review Board for Animal Research of the Shanghai Tenth People's Hospital of Tongji University (SHDSYY-2018-3912).

Dextran sulfate sodium (DSS)-induced colitis model in mice
DSS-induced colitis model in mice was established as described previously. 13 Briefly, WT and Cd177 −/ − mice were treated with 2% DSS in drinking water for 7 days, then replaced with sterile water for another 3 days. On day 10, all mice were sacrificed. The severity of colitis was scored daily by recording standard parameters including body weight, diarrhea, bloody stool, and survival rates. Colon tissues were removed, fixed in 10% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Histological scores were calculated by combining the scores for each of six parameters, including the degree of inflammation in lamina propria (0-none, 1-mild, 2-moderate, 3-severe), goblet cell loss (0-none, 1-mild/ moderate, 2-severe), disrupted crypts (0-normal, 1-hyperplastic, 2-disorganization, 3-crypt loss), presence of crypt abscesses (0-absent, 1-present), mucosal erosion (0-absent, 1-present) and submucosal ulceration spread to transmural involvement (0-none, 1-submucosal, 2-transmural), for a maximum score of 12. Three to four worst regions were selected, scored, and then averaged to determine the final score.

Isolation of intestinal IELs and lamina propria mononuclear cells
As described previously, 14 the intestines were carefully cleaned from the mesentery and flushed of the fecal contents. Intestines were opened longitudinally, cut into 0.5-1.0 cm pieces, and then washed with cold phosphate-buffered saline (PBS) to remove the fecal contents. After the digestion by 1 mM EDTA in PBS at 37°C for 2 × 20 minutes, primary IELs were collected in the supernatants and further purified via density gradient centrifugation with 40% and 75% Percoll-Roswell Park Memorial Institute (RPMI) solution. Colon tissues were then digested by collagenase A (1 mg/mL; Sigma-Aldrich, St. Louis, Missouri, USA) at 37°C for 30 minutes. The single-cell suspension was collected and further purified via density gradient centrifugation with 40% and 75% Percoll-RPMI solution. Lamina propria mononuclear cells (LPMCs) were then collected from the interface and suspended in 10% fetal bovine serum (FBS)-RPMI medium.

Flow cytometric analysis
For cell surface staining, IELs and LPMCs were obtained from Cd177 -/mice and WT littermates, first incubated with Fc Block (BD Biosciences), and then stained with fluorochrome-conjugated mAbs against TCRγ/δ, TCRβ, CD4, CD8a and CD8b.2, respectively, for 30 minutes at 4°C. 15 Concomitantly, the Live/Dead Fixable Dead Cell stain kits (Invitrogen, Eugene, OR) was used to exclude dead cells. For intracellular cytokine staining, IELs were treated with phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; Sigma-Aldrich) and ionomycin (750 ng/mL; Sigma-Aldrich) for 5 hours at 37°C, along with the stimulation of brefeldin A (3 μg/mL; eBioscience) for the last 3 hours. Subsequently, these cells were harvested and processed for surface staining, followed by fixation and permeabilization for 30 minutes at 4°C. After three washes, intracellular staining was performed with fluorochrome-conjugated anti-IFN-γ, anti-IL-17A, anti-TNF-α, and anti-IL-10 mAbs. All stained samples were analyzed on a BD FACS Canto II Flow Cytometer. All data were processed using FlowJo software (Version 10.0.7, Tree Star; Ashland, Oregon, USA).

Depletion of neutrophils in vivo
To delete neutrophils, mice were injected intraperitoneally at a dose of 100 μg of anti-mouse Ly6G antibody (clone 1A8, Bio X Cell; West Lebanon, New Hampshire, USA) in PBS on days 0, 3, 6 and 9 before DSS administration. Rat IgG2a (clone 2A3, Bio X cell) was used as isotype control. 16

Bacterial infection of mice
Citrobacter rodentium (CR) strain DBS100 (ATCC51459; American Type Culture Collection, Rockefeller, Maryland, USA) was generously provided by Dr. Youcun Qian (The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China). CR were prepared by gently shaking bacteria overnight at 37°C in Luria-Bertani broth. Bacterial cultures were progressively diluted and plated on MacConkey agar plates to obtain the optimal loads of CFUs administered. For infection experiments, mice were fast for 8 hours before oral inoculation with 2 × 10 9 CFUs of CR in a total volume of 100 μl per mouse. Mortality was monitored daily throughout the infection. The changes in body weights were assessed at the beginning of infection and every 2 days after infection. 17

Antibiotics administration
To deplete gut bacteria of SPF mice, quadruple antibiotics, including ampicillin (1 g/L; Sigma-Aldrich), metronidazole (1 g/L; Sigma-Aldrich), neomycin (1 g/L; Sigma Aldrich), and vancomycin (0.5 g/L; Wako), were mixed in sterile drinking water for 4 weeks as described previously. 18 Antibiotics cocktails were given to mice continuously until sacrificed during the experiments.

DMF treatment
Mice were given DMF (100 mg/kg) by oral gavage daily in an emulsion of 0.6% methocel from 0 to 10 days of DSS treatment. An equivalent volume of 0.6% methocel vehicle was served as control.

Single-cell RNA sequencing (scRNA-seq) analysis
Single-cell suspensions were isolated from the mouse colon epithelial layer through a positive selection of CD45 + immuno-magnetic beads and underwent cell viability detection. Samples with viabilities >85% were available for scRNA-seq analysis based on NovaSeq6000 platform, and sequencing libraries were generated with BD Rhapsody protocol adapted from the BD Resolve system as described previously. 19 In brief, single cells were captured into >200,000 microwells with cell barcodes and unique molecular identifier barcodes before cell lysis, and mRNA capture via ligation of poly A. Cells with mitochondrial RNAs >40% and expression of fewer than 200 genes were discarded. PCA and tSNE analysis were used for the single-cell-to-cell relation description. Graphcluster and K-mean were utilized for cell clustering, and the Wilcox rank-sum test was used for marker gene analysis. Gene ontology (GO) analysis was annotated from NCBI, UniProt, and Gene Ontology. Fisher's exact test was applied to identify the significant GO categories, and FDR was used to correct the p values.

Statistical analysis
All of the data were expressed as mean ± SEM and analyzed using GraphPad Prism 8. Two-group comparisons were performed using Student's t-test. Multiple sample comparisons were performed using the Mann-Whitney test. Two-group comparisons for data with different treatments were analyzed and were performed using two-way ANOVAs with Tukey's multiple comparisons test. *p < .05, **p < .01, ***p < .001 and ****p < .0001 were considered statistically significant.

Neutrophils modulate IEL properties during gut inflammation
To investigate the role of neutrophils in modulating the IEL response, we first established an experimental model of acute colitis in WT mice induced by 2% DSS and concomitantly treated intraperitoneally with anti-Ly6G antibody to deplete neutrophils in vivo (Supplementary Figure S1a). Neutrophil-depleted mice developed more severe colitis than controls, characterized by significant weight loss, aggravated histological lesions, impaired mucosal barrier function, and expression of proinflammatory cytokines (e.g., IL-1β, IL-17A, IFN-γ and TNF-α) in the colon tissues (Supplementary Figure S1b Figure S1g). However, we found that TCRγδ + CD8αα + IELs increased in the colon of colitic mice, which were further upregulated in neutrophil-depleted colitic mice compared with controls ( Figure 1a). TCRγδ + CD8αα + IELs are largely exhausted during chronic inflammation, which are mainly considered a guard in the intestinal mucosa and contribute to pathogen restriction and tight regulation of innate and adaptive immune responses. 20 TCRγδ + cells expressing high gut-homing integrins (CD103 and α4β7) are considered 'inflammatory' cells due to the enhanced Th1/Th17 cell differentiation after the adoptive transfer. 21 To investigate the properties of TCRγδ + CD8αα + IELs during colitis, we examined the expression of different inflammatory cytokines by flow cytometry. Consistent with more severe colitis in neutrophil-depleted mice, higher proportions of IFN-γ-, IL-17A-and TNF-αexpressing TCRγδ + CD8αα + IELs were detected in these mice compared to WT controls ( Figure 1c). In contrast, TCRαβ + CD4 +· IELs were found to be decreased in the colon of neutrophil-depleted mice during DSS-induced acute colitis ( Figure 1b) and produced low levels of IL-10 ( Figure 1c). Additionally, there were no consistent changes in the proportions and counts of TCRαβ + CD8αα + and TCRαβ + CD8αβ + IELs among these groups (Supplementary Figure S1g). Collectively, these data reveal that expanded TCRγδ + CD8αα + IELs manifest with proinflammatory properties after neutrophil depletion, suggesting that neutrophils modulate IEL functions and maintain gut homeostasis during intestinal mucosal inflammation.

TCRγδ + CD8αα + IELs expand in colitic Cd177 −/− mice
Neutrophils have been shown to regulate the innate immune response at the early stage of inflammation. 22 Our previous studies demonstrated that CD177 + neutrophils function as an activated subset of neutrophils with high bactericidal capacity, increased expression of IL-22 and TGF-β, and decreased release of proinflammatory mediators such as IFN-γ, IL-6, and IL-17A. 4 Hence, we reasoned whether CD177 + neutrophils might predominantly modulate the expansion of TCRγδ + CD8αα + IELs under inflammatory conditions. To this end, we induced acute colitis in Cd177 −/− and WT mice with DSS insults and simultaneously depleted neutrophils with anti-Ly6G antibody intraperitoneally. We found aggravated mucosal damage and architectural distortion in the intestines of colitic Cd177 −/− mice compared with WT controls, as manifested by aggravated histological lesions and damaged mucosal barrier (Figure 1d,e), indicating that Cd177 −/− mice developed more severe colitis under DSS insults. Our previous studies have demonstrated no differences in immune cells in LPMC (such as CD4 + , CD8 + T cells, and B cells) between Cd177 −/− and WT mice under steady or inflammatory conditions, 4 and we further observed no significant changes in the compositions of IELs isolated from the colon at the steady-state (Supplementary Figure S2a). Interestingly, we found that TCRγδ + CD8αα + IELs were also increased, while TCRαβ + CD4 +· IELs were decreased in the colon of the colitic Cd177 −/− mice (Figure 1f,g). TCRγδ + CD8αα + IELs in the colon of Cd177 −/− mice also produced more IFN-γ,·IL-17A, and TNF-α than WT controls (Figure 1h). However, depletion of total neutrophils using anti-Ly6G antibody did not further affect the frequencies and cytokine production (e.g., IFN-γ,·IL-17A, and TNF-α) of TCRγδ + CD8αα + IELs in colitic Cd177 −/− mice compared with those in control IgG2a-treated Cd177 −/− mice, indicating that CD177 + neutrophils mainly regulate TCRγδ + CD8αα + IELs during colitis (Supplementary Figure S2b,c and Figure 1f).
As a complementary approach, we orally infected WT and Cd177 −/− mice with Citrobacter rodentium (CR, 2 × 10 9 CFUs/mouse), and found that Cd177 −/− mice developed more severe colitis with more weight loss, impaired epithelial barrier integrity than WT controls after CR infection (Supplementary Figure S3a-c). Consistent with the results from DSS-induced colitis, exposure to CR infection also caused an increase in TCRγδ + CD8αα + IELs but a decrease in TCRαβ + CD4 +· IELs in the colon of Cd177 −/− mice compared with WT controls (Supplementary Figure S3d,e). Taken together, these data demonstrated that CD177 + neutrophils control the colitogenic TCRγδ + CD8αα + IEL expansion during colitis.
To assess whether the microbiota was associated with the changes in TCRγδ + CD8αα + IELs when neutrophils were depleted, we analyzed scRNAseq data related to microbial responses of TCRγδ + CD8αα + IELs. We observed distinct gene expression profiles in TCRγδ + CD8αα + IELs from colitic Cd177 −/− mice, which were preferentially involved in bacterial invasion of the epithelial cells (74 genes including Rhog, Arpc1a, Rac1, Pik3cb, and Arpc2), defense response to Gramnegative bacteria (122 genes including Lypd8, double positive IELs) in the colon of the indicated groups on day 10. Bar charts showed the proportion and the absolute number of indicated IELs. (c) The frequencies of IFN-γ-, IL-17A-, TNF-α-, and IL-10-expressing TCRγδ + CD8αα + IELs and TCRαβ + CD4 + IELs isolated from the colon of the indicated WT mice (n = 6 per group) were detected by flow cytometry and counted into the statistical chart. (d-h) Acute colitis was induced in WT and Cd177 −/− mice (n = 6 per group) by 2% DSS in drinking water and treated intraperitoneally with αLy6G antibody and rat IgG2a (100 μg/mouse), respectively, every three days. (d) Representative H&E staining of the colon tissues from WT and Cd177 −/− mice treated with rat IgG2a or αLy6G antibody throughout the 10-day observation after DSS insults. Scale bar, 100 µm. Histological scores were shown as indicated (n = 6 per group). (e) Serum levels of FITC-dextran (4 kD, 600 mg/kg) in each group. (f, g) Flow cytometric analysis of TCRγδ + CD8αα + IELs (f) and TCRαβ + CD4 + IELs (g, gated TCRαβ + IELs) in the colon of each group. Bar charts showed the proportion and the absolute number of indicated IELs. (h) The frequencies of IFN-γ-, IL-17A-, TNF-α-, and IL-10expressing TCRγδ + CD8αα + IELs isolated from the colon of colitic WT and Cd177 −/− mice treated with rat IgG2a or αLy6G antibody (n = 6 per group), assessed by flow cytometry and counted into the statistical chart. Data were representative of three independent experiments. *p < .05; **p < .01; ***p < .001; and ****p < .0001 and ns, no significant difference.
After determining the association between IEL changes and GSDMD-induced pyroptosis based on single-cell sequencing data, we then comprehensively analyzed the differential metabolites through microbial metabolomics data and screened out the differential expressed metabolites associated with regulation of pyroptosis in order to clarify the underlying mechanisms. Among these metabolites, DMF as a derivative of fumarate, a metabolic intermediate in the tricarboxylic acid (TCA) cycle converted from succinate by the enzyme succinate dehydrogenase, was markedly decreased in the fecal samples of colitic Cd177 −/− mice (Figure 4d, e). DMF has shown broad anti-inflammatory effects on various immune cells, including neutrophils, dendritic cells, macrophages, NK, B, and T cells, and has already been used to treat patients with multiple sclerosis and psoriasis. 32 Treatment with DMF decreases peripheral T cell counts, especially CD8 + T cells, in psoriasis patients through increased apoptosis and declined proliferation. 33 Moreover, administration of DMF has been demonstrated to alleviate mucosal inflammation in the DNBSinduced murine colitis model. 34 DMF-induced protein succination suppresses cleaved-gasdermin D (GSDMD)-mediated pyroptosis. 35 Consistent with the decreased levels of DMF in the feces of colitic Cd177 −/− mice, fumarate-producing bacteria (including Akkermansia, Coriobacteriaceae, Erysipelotrichaceae, and Peptostreptococcus) were decreased, 36,37 while fumarate-consuming bacteria (including Bacteroides, Clostridium, Acinetobacter, Vibrio, Propionibacterium, Prevotella, Clostridiales, Ruminococcus, Geobacter, Desulfovibrio and Pseudomonas) were increased in colitic Cd177 −/− mice (Figure 4f), [38][39][40][41][42][43][44] suggesting that the altered gut microbiota was responsible for the decrease of DMF in the fecal samples of these Cd177 −/− mice. Collectively, these data provide a hypothesis that microbial metabolite DMF may be associated with the regulation of IEL function and maintain intestinal mucosal homeostasis, and that deficiency of DMF may trigger intestinal mucosal inflammation and induce fundamental activation of pathogenic TCRγδ + CD8αα + IELs.
It has been shown that GSDMD expression is increased in inflamed mucosa of IBD patients by scRNA-seq analysis. 45 Notably, we observed a significant increase in Gsdmd expression in TCRγδ + CD8αα + IELs of colitic Cd177 −/− mice compared with WT controls (Figure 2e). The poreforming GSDMD-N was markedly increased in TCRγδ + CD8αα + IELs of colitic Cd177 −/− mice, as well as significantly higher expression of IL-1β and IL-18 (Figure 5e,f), suggesting that Cd177 −/− TCRγδ + CD8αα + IELs were more prone to pyroptotic cell death. However, DMF administration protected against TCRγδ + CD8αα + IEL pyroptosis, as evidenced by reduced expression of GSDMD-N, IL-1β and IL-18 (Figure 5e,f). Consistent with these findings, higher levels of GSDMD-N were also detected in IECs and colon tissues of Cd177 −/− mice after DSS insults but concomitantly blocked by DMF (Supplementary Figure S8f). Collectively, these data indicate that a decrease in microbial metabolite DMF due to the dysregulated immunoregulation of neutrophils on gut microbiota causes TCRγδ + CD8αα + IEL expansion.

Discussion
Intestinal resident IELs and dynamically circulating neutrophils are potent host defenders against gut pathogens due to the early response and robust cytotoxic effects. Their inappropriate functional status is associated with intestinal inflammatory diseases, such as IBD. 9,46 Emerging evidence establishes the crucial role of neutrophils in the pathogenesis of IBD as a Janus-faced modulator relevant to  The mRNA levels of pyroptosis-associated cytokines in flow-sorted TCRγδ + CD8αα + IELs (n = 6 per group). Data were representative of three independent experiments. *p < .05; **p < .01; ***p < .001; and ****p < .0001 and ns, no significant difference. aggravated intestinal inflammation and inflammation resolution gut mucosa in IBD, paradoxically. 46 However, how neutrophils regulate IEL functions in the pathogenesis of IBD remains elusive, and the underlying mechanisms whereby neutrophils regulate the IELs in the context of a massive and diverse microbiota are not well defined. Our previous study demonstrated that CD177 + neutrophils play a protective role in IBD through increased bactericidal activity and IL-22 production. 4 However, we found no significant difference in IL-22 levels from the colon between colitic Cd177 −/− mice and WT littermates, thus we speculated that IL-22 does not affect IEL activation and expansion. Our study uncovered the unknown mechanisms by which neutrophils regulate IEL function through modulating gut microbiota and their metabolite DMF, which controls the expansion of colitogenic TCRγδ + CD8αα + IELs. TCRγδ + CD8αα + IELs have proinflammatory functional heterogeneity with an overarching integrated feature of increased GSDMD-mediated pyroptosis during intestinal inflammation. With an increase of antimicrobial peptides (e.g., s100a8, s100a9, and Reg3b) and defense response-related genes (e.g., Gzma, Gzmb, Klrb1b, and Fasl), TCRγδ + CD8αα + IELs appear protective bactericidal activity and immunoregulatory potential in an appropriate activated state, while the overactivation causes the compromised mucosal homeostasis and aggravated inflammatory response, leading to severe tissue damage in the gut mucosa.
Gut microbiota has been found to be associated with TCRγδ + ·IEL activation and cytokine interactions and is crucial in shaping the IEL compartment. 47 Our study demonstrated that microbial homeostasis under the control of neutrophils enables fine-tuning the delicate balance between the protective and pathogenic characters of TCRγδ + CD8αα + IELs, thus providing novel insights into how neutrophils modulate IEL immune response through the alterations in microbial metabolites, particularly a decline of DMF. Consistently, supplementation of DMF unequivocally alleviated experimental colitis and restrained the overactivation of TCRγδ + CD8αα + IELs in colitic Cd177 −/− mice. Additionally, deletion of neutrophil leads to dysbiosis in gut microbiota, and an overgrowth of invasive microbiota may also cross the barrier directly into contact with the epithelial and immune cells and activate TCRγδ + CD8αα + IELs. From a broader perspective, DMF can also prevent the pyroptosis of IECs and even the entire intestinal tissue, which is not limited exclusively to immunoregulatory effects on immune cells in the intestinal tissue. 34 Given that the maintenance of intestinal homeostasis requires the synergy of multiple immune cells, the interactions between IECs and IELs may also play an important role in maintaining the immune homeostasis and inflammatory response in gut mucosa. Thus, these findings illustrate an important role of DMF in regulating intestinal inflammation and maintaining gut immune homeostasis. Currently, the FDA has approved DMF (e.g., Tecfidera) to treat multiple sclerosis and psoriasis, but DMF can also induce lymphopenia and restrict glycolysis in lymphocytes. 48 Therefore, clinical trials with DMF may be warranted to assess its efficacy and side-effects in the treatment of IBD patients. However, DMF has not yet been marketed in China, and we are looking forward to the opportunity to conduct clinical trials of DMF in IBD in the future.
Given the loss of a rapid, effective response against exogenous pathogens by neutrophils, early defense against pathogens that invade the intestine requires the innate immune cells of the intestinal immunity to take over. In the complex and delicate intestinal immune network where mucus, immune cells, and symbiotic flora act together, there is a delineated way for the microbiota to regulate immune cells through metabolites. Based on the fact that there are many patients with neutropenia and a series of complications, such as fever, infection, diarrhea, and compromised mucosal homeostasis in the gastrointestinal tract, it is essential to better understand the immune response and self-regulation system in the absence of neutrophils. In patients with intestinal immunopathology, abdominal pain, diarrhea, and hematochezia seriously lessen the quality of life. Hence, it is considered to understand and improve intestinal immunity at the onset of neutropenia. IELs, as the first line of defense in the intestinal immune system, are also affected and altered during the process of neutrophil disability. We found that DMF derived from microbial metabolites could regulate IELs, thus providing a novel clue for maintaining intestinal immune homeostasis. Collectively, our study shed some light on seeking promising therapies for neutropenia in patients suffering from systematic autoimmune diseases (e.g., systemic lupus erythematosus), immune-related disorders in the gastrointestinal tract, or receiving an immunosuppressant (e.g., azathioprine). Therefore, such a strategy to restore gut microbiota-derived metabolites or restrict TCRγδ + CD8αα + IEL overactivation will provide an avenue for potential advanced treatment.